This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Copper (Cu) is an essential trace element for most organisms. However, when in excess Cu is also toxic, mainly caused by its redox properties and the generation of radicals. Studies using metallic Cu surfaces demonstrated excellent antimicrobial efficacy against a wide variety of bacteria. Cu surfaces killed bacteria within a few minutes. Nevertheless, the cellular targets of Cu toxicity remain obscure. Also, no studies addressed the lethal effects of Cu surfaces towards eukaryotes such s fungi. We will investigate the antimicrobial properties of metallic Cu surfaces against pro- and eukaryotes. This will be the basis for harnessing those properties in the battle against the spread of pathogens in hygiene sensitive areas like hospitals. This pilot project will explore two specific aims: Aim 1: Identify the molecular targets that govern inactivation of bacteria and fungi on metallic Cu surfaces. Use of analytical methods such as Inductively-Coupled-Plasma-Mass-Spectrometry or intracellular metal-responsive dyes will reveal if organisms exposed to Cu surfaces indeed accumulate toxic levels of Cu ions or if rather indirect effects such as redox-stress is the underlying mechanism of kill. Also, application of Atomic-Force-Microscopy and Epifluorescence Microscopy will indicate whether cells exposed to metallic Cu are inactivated because their membranes suffered lethal damage. Analysis of mutant cells lacking diverse oxidative stress response systems will clarify the role of reactive oxygen species in Cu surface mediated killing. Finally, it is currently controversially discussed if Cu toxicity targets genetic material within the cell. The response of an intracellular reporter system that monitors the integrity of the chromosome will disclose if lethal DNA damage is the underlying mechanism of action of Cu toxicity. Aim 2: Identify genetic factors responsible for resistance against metallic Cu surfaces. It is likely that bacteria that are constantly exposed to metallic Cu surfaces have evolved specific resistance mechanisms. Recently, we have isolated resistant bacteria from Cu coins. As part of the pilot project a genetic approach is planned that will elucidate the presence of plasmids conferring resistance against Cu surfaces. Genes encoding transferable resistances will be sequenced and characterized by transposon mutagenesis and mutant analysis. Further, comparison of the co-occurrence of Cu and antibiotics resistance will clarify if there is an intrinsic connection between these traits. Knowledge on the genetics and biochemistry of teh Cu detoxification systems of the coinage isolates will significantly contribute to the understanding of the mode of action of metallic Cu surfaces. On the long term, this will likely be valuable for developing advanced strategies to improve Cu surfaces that are also competent to inactivate these bacteria.